Detection of analytes using reorganization energy

ABSTRACT

The invention relates to novel methods and compositions for the detection of analytes using the nuclear reorganization energy, λ, of an electron transfer process

[0001] This application is a continuing application of U.S. Ser. No.08/873,977, filed Jun. 12, 1997.

FIELD OF THE INVENTION

[0002] The invention relates to novel methods and compositions for thedetection of analytes based on changes in the nuclear reorganizationenergy, λ, of electron transfer process.

BACKGROUND OF THE INVENTION

[0003] Electron transfer reactions are crucial steps in a variety ofbiological transformations ranging from photosynthesis to aerobicrespiration. Studies of electron transfer reactions in both chemical andbiological systems have led to the development of a large body ofknowledge and a strong theoretical base, which describes the rate ofelectron transfer in terms of a definable set of parameters.

[0004] Electronic tunneling in proteins and other biological moleculesoccurs in reactions where the electronic interaction of the redoxcenters is relatively weak. Semiclassical theory reaction predicts thatthe reaction rate for electron transfer depends on the driving force(−ΔG°), a nuclear reorganization parameter (λ), and theelectronic-coupling strength (H_(AB)) between the reactants and productsat the transition state, according to the following equation:

k _(ET)=(4π³ /h ² λk _(B) T)^(½)(H _(AB))²exp[(−ΔG°+λ)² /λk _(B) T]

[0005] The nuclear reorginzation energy, λ, in the equation above isdefined as the energy of the reactants at the equilibrium nuclearconfiguration of the products. There are two components of λ; “outersphere” effects (λ₀) and “inner sphere” effects (λ₁). For electrontransfer reactions in polar solvents, the dominant contribution to λarises from the reorientation of solvent molecules in response to thechange in charge distribution of the reactants. The second component ofλ comes from the changes in bond lengths and angles due to changes inthe oxidation state of the donors and acceptors.

[0006] It is an object of the present invention to provide methods forthe detection of target analytes exploiting changes in the solventreorganization energy of electron transfer reactions.

SUMMARY OF THE INVENTION

[0007] In accordance with the above objects, the present inventionprovides methods of detecting a target analyte in a test sample. Themethod comprises binding an analyte to a redoxactive complex. The redoxactive complex comprises a solvent accessible transition metal complexhaving at least one coordination site occupied by a polar coordinationgroup and a binding ligand which will bind the target analyte. Thecomplex is bound to an electrode. Upon binding, a solvent inhibitedtransition metal complex is formed and electron transfer is detectedbetween the solvent inhibited transition metal complex and theelectrode. The methods also include applying at least a first inputsignal to the solvent inhibited transition metal complex.

[0008] In a further aspect, the invention provides methods of detectinga target analyte in a test sample comprising associating an analyte witha redox active complex. The redox active complex comprises a solventinhibited transition metal complex, and a binding ligand which will bindthe target analyte. Upon association, a solvent accessible transitionmetal complex is formed, which is then detected.

[0009] In an additional aspect, the invention provides methods ofdetecting a target analyte in a test sample comprising associating ananalyte with a redox active complex. The complex comprises a solventinhibited transition metal complex, a binding ligand which will bind thetarget analyte, and an analyte analog. The complex is bound to anelectrode, and upon association, a solvent accessible transition metalcomplex is formed, which is then detected.

[0010] In a further aspect, the invention provides compositionscomprising an electrode with a covalently attached redox active complex.The complex comprises a binding ligand and a solvent accessible redoxactive molecule, which has at least one, and preferably two or threecoordination sites occupied by a polar coordination group, one or moreof which may be a water molecule.

[0011] In a further aspect, the present invention provides an apparatusfor the detection of target analytes in a test sample, comprising a testchamber comprising a first and a second measuring electrode. The firstmeasuring electrode comprises a covalently attached redox active complexcomprising a solvent accessible transition metal complex, preferablyhaving at least three coordination sites occupied by a polarcoordination group, and a binding ligand. The apparatus furthercomprises an AC/DC voltage source electrically connected to the testchamber, and an optional signal processor for detection.

DETAILED DESCRIPTION

[0012] The present invention provides methods and compositions for thedetection of target analytes using changes in the solvent reorganizationenergy of transition metal complexes upon binding of the analytes, tofacilatate electron transfer between the transition metal complex and anelectrode. This invention is based on the fact that a change in theoxidation state of a redox active molecule such as a transition metalion, i.e. upon the acceptance or donation of an electron, results in achange in the charge and size of the metal ion. This change in thecharge and size requires that the surrounding solvent reorganize, tovarying degrees, upon this change in the oxidation state.

[0013] For the purposes of this invention, the solvent reorganizationenergy will be treated as the dominating component of λ. Thus, if thesolvent reorganization energy is high, a change in the oxidation statewill be impeded, even under otherwise favorable conditions.

[0014] In conventional methodologies using electron transfer, thissolvent effect is minimized by using transition metal complexes thatminimize solvent reorganization at the redox center, generally by usingseveral large hydrophobic ligands which serve to exclude water. Thus,the ligand for the transition metal ions traditionally used arenon-polar and are generally hydrophobic, frequently containing organicrings.

[0015] However, the present invention relies on the novel idea ofexploiting this solvent reorganization energy to serve as the basis ofan assay for target analytes. In the present invention, transition metalcomplexes that are solvent accessible, i.e. have at least one, andpreferably more, small, polar ligands, and thus high solventreorganization energies, are used. Thus, at initiation energies lessthan the solvent reorganization energy, no significant electron transferoccurs. However, upon binding of a generally large target analyte, thetransition metal complexes becomes solvent inhibited, inaccessible topolar solvents generally through steric effects, which allows electrontransfer at previously inoperative initiation energies.

[0016] Thus, the change in a transition metal complex from solventaccessible to solvent inhibited serves as a switch or trigger forelectron transfer. This thus becomes the basis of an assay for ananalyte. Closs and Miller have shown that there is a decrease in lambdain nonpolar solvents in their work on Donor(bridge)Acceptor electrontransfer reactions in solution. (Closs and Miller, Science, 240,440-447, (1988). This idea also finds conceptual basis in work done withmetmyoglobin, which contains a coordinated water molecule in thehexacoordinate heme iron site and does not undergo self-exchange veryrapidly (rate constant k₂₂ 1M⁻¹s⁻¹). Upon chemical modification, theheme becomes pentacoordinate, removing the water, and the self-exchangerate constant increases significantly (rate constant k₂₂ 1×10⁴ M⁻¹s⁻¹);see Tsukahara, J. Am. Chem. Soc. 111:2040 (1989).

[0017] Without being bound by theory, there are two general mechanismswhich may be exploited in the present invention. In a preferredembodiment, the binding of a target analyte to a binding ligand which issterically to a solvent accessible transition metal complex causes oneor more of the small, polar ligands on the solvent accessible transitionmetal complex to be replaced by one or more coordination atoms suppliedby the target analyte, causing a decrease in the solvent reorganizationenergy for at least two reasons. First, the exchange of a small, polarligand for a generally larger, nonpolar ligand that will generallyexclude more water from the metal, lowering the required solventreorganization energy (i.e. an inner sphere λ, effect). Secondly, theproximity of a generally large target analyte to the relatively smallredox active molecule will sterically exclude water within the first orsecond coordination sphere of the metal ion, also decreasing the solventreorganization energy.

[0018] Alternatively, a preferred embodiment does not necessarilyrequire the exchange of the polar ligands on the metal ion by a targetanalyte coordination atom. Rather, in this embodiment, the polar ligandsare effectively irreversibly bound to the metal ion, and the decrease insolvent reorganization energy is obtained as a result of the exclusionof water in the first or second coordination sphere of the metal ion asa result of the binding of the target analyte; essentially the water isexcluded (i.e. an outher sphere λ_(o) effect).

[0019] Accordingly, the present invention provides methods for thedetection of target analytes. The methods generally comprise binding ananalyte to a binding ligand that is either associated with (forming aredox active complex) or near to a transition metal complex. Thetransition metal complex is bound to an electrode generally through theuse of a conductive oligomer. Upon analyte binding, the reorganizationenergy of the transition metal complex decreases to form a solventinhibited transition metal complex, to allow greater electron transferbetween the solvent inhibited transition metal complex and theelectrode.

[0020] Accordingly, the present invention provides methods for thedetection of target analytes. By “target analyte” or “analyte” orgrammatical equivalents herein is meant any molecule, compound orparticle to be detected. As outlined below, target analytes preferablybind to binding ligands, as is more fully described below

[0021] Suitable analytes include organic and inorganic molecules,including biomolecules. In a preferred embodiment, the analyte may be anenvironmental pollutant (including pesticides, insecticides, toxins,etc.); a chemical (including solvents, polymers, organic materials,etc.); therapeutic molecules (including therapeutic and abused drugs,antibiotics, etc.); biomolecules (including hormones, cytokines,proteins, lipids, carbohydrates, cellular membrane antigens andreceptors (neural, hormonal, nutrient, and cell surface receptors) ortheir ligands, etc); whole cells (including procaryotic (such aspathogenic bacteria) and eucaryotic cells, including mammalian tumorcells); viruses (includin etroviruses, herpesviruses, adenoviruses,lentiviruses, etc.); and spores; etc. Particularly preferred analytesare environmental pollutants; nucleic acids; proteins (includingenzymes, antibodies, antigens, growth factors, cytokines, etc);therapeutic and abused drugs; cells; and viruses.

[0022] By “nucleic acid” or “oligonucleotide” or grammatical equivalentsherein means at least two nucleotides covalently linked together. Anucleic acid of the present invention will generally containphosphodiester bonds, although in some cases, as outlined below, anucleic acid analogs are included that may have alternate backbones,comprising, for example, phosphoramide (Beaucage et al., Tetrahedron49(10):1925 (1993) and references therein; Letsinger, J. Org. Chem.35:3800 (1970); Sprinzl et al., Eur. J. Biochem. 81:579 (1977);Letsinger et al., Nucl. Acids Res. 14:3487 (1986); Sawai et al, Chem.Lett. 805 (1984), Letsinger et al., J. Am. Chem. Soc. 110:4470 (1988);and Pauwels et al., Chemica Scripta 26:141 91986)), phosphorothioate,phosphorodithioate, O-methylphophoroamidite linkages (see Eckstein,Oligonucleotides and Analogues: A Practical Approach, Oxford UniversityPress), and peptide nucleic acid backbones and linkages (see Egholm, J.Am. Chem Soc. 114:1895 (1992); Meier et al., Chem. Int. Ed. Engl.31:1008 (1992); Nielsen, Nature, 365:566 (1993); Carlsson et al., Nature380:207 (1996), all of which are incorporated by reference). Nucleicacids containing one or more carbocyclic sugars are also included withinthe definition of nucleic acids (see Jenkins et al., Chem. Soc. Rev.(1995) pp169-176). These modifications of the ribose-phosphate backbonemay be done to facilitate the addition of moieties, or to increase thestability and half-life of such molecules in physiological environments.

[0023] The nucleic acids may be single stranded or double stranded, asspecified, or contain portions of both double stranded or singlestranded sequence. The nucleic acid may be DNA, both genomic and cDNA,RNA or a hybrid, where the nucleic acid contains any combination ofdeoxyribo- and ribo-nucleotides, and any combination of bases, includinguracil, adenine, thymine, cytosine, guanine, inosine, xathanine andhypoxathanine, etc. As used herein, the term “nucleoside” includesnucleotides, and modified nucleosides such as amino or thio modifiednucleosides.

[0024] By “proteins” or grammatical equivalents herein is meantproteins, oligopeptides and peptides, and analogs, including proteinscontaining non-naturally occuring amino acids and amino acid analogs,and peptidomimetic structures.

[0025] As will be appreciated by those in the art, a large number ofanalytes may be detected using the present methods; basically, anytarget analyte for which a binding ligand, described below, may be mademay be detected using the methods of the invention.

[0026] In a preferred embodiment, the target analyte is added orintroduced to a redox active complex, which is preferably attached to anelectrode. By “redox active complex” herein is meant a complexcomprising at least one transition metal complex and at least onebinding ligand, which, as more fully described below, may be associatedin a number of different ways. By “transition metal complex” or “redoxactive molecule” or “electron transfer moiety” herein is meant ametal-containing compound which is capable of reversibly orsemi-reversibly transfering one or more electrons. It is to beunderstood that electron donor and acceptor capabilities are relative;that is, a molecule which can lose an electron under certainexperimental conditions will be able to accept an electron underdifferent experimental conditions. It is to be understood that thenumber of possible transition metal complexes is very large, and thatone skilled in the art of electron transfer compounds will be able toutilize a number of compounds in the present invention. Transitionmetals are those whose atoms have a partial or complete d shell ofelectrons. Suitable transition metals for use in the invention include,but are not limited to, cadmium (Cd), copper (Cu), cobalt (Co),palladium (Pd), zinc (Zn), iron (Fe), ruthenium (Ru), rhodium (Rh),osmium (Os), rhenium (Re), platinium (Pt), scandium (Sc), titanium (Ti),Vanadium (V), chromium (Cr), manganese (Mn), nickel (Ni), Molybdenum(Mo), technetium (Tc), tungsten (W), and iridium (Ir). That is, thefirst series of transition metals, the platinum metals (Ru, Rh, Pd, Os,Ir and Pt), along with Fe, Re, W, Mo and Tc, are preferred. Particularlypreferred are metals that do not change the number of coordination sitesupon a change in oxidation state, including ruthenium, osmium, iron,platinium and palladium, with ruthenium and iron being especiallypreferred. Generally, transition metals are depicted herein as M.

[0027] The transition metal ions are complexed with ligands that serveto provide the coordination atoms for the binding of the metal ion.Generally, it is the composition or characteristics of the ligands thatdetermine whether a transition metal complex is solvent accessible. By“solvent accessible transition metal complex” or grammatical equivalentsherein is meant a transition metal complex that has at least one,preferably two, and more preferably three, four or more small polarligands. The actual number of polar ligands will depend on thecoordination number (n) of the metal ion. Preferred numbers of polarligands are (n-1) and (n-2). For example, for hexacoordinate metals,such as Fe, Ru, and Os, solvent accessible transition metal complexespreferably have one to five small polar ligands, with two to five beingpreferred, and three to five being particularly preferred, depending onthe requirement for the other sites, as is more fully described below.Tetracoordinate metals such as Pt and Pd preferably have one, two orthree small polar ligands.

[0028] It should be understood that “solvent accessible and solventinhibited” are relative terms. That is, at high applied energy, even asolvent accessible transition metal complex may be induced to transferan electron.

[0029] The other coordination sites of the metal are used for attachmentof the transition metal complex to either a binding ligand (directly orindirectly using a linker), to form a redox active complex, or to theelectrode (frequently using a spacer, as is more fully described below),or both. Thus for example, when the transition metal complex is directlyjoined to a binding ligand, one, two or more of the coordination sitesof the metal ion may be occupied by coordination atoms supplied by thebinding ligand (or by the linker, if indirectly joined). In addition, oralternatively, one or more of the coordination sites of the metal ionmay be occupied by a spacer used to attach the transition metal complexto the electrode. For example, when the transition metal complex isattached to the electrode separately from the binding ligand as is morefully described below, all of the coordination sites of the metal (n)except 1 (n-1) may contain polar ligands.

[0030] Suitable small polar ligands, generally depicted herein as “L”,fall into two general categories, as is more fully described below. Inone embodiment, the small polar ligands will be effectively irreversiblybound to the metal ion, due to their characteristics as generally poorleaving groups or as good sigma donors, and the identity of the metal.These ligands may be referred to as “substitutionally inert”.Alternatively, as is more fully described below, the small polar ligandsmay be reversibly bound to the metal ion, such that upon binding of atarget analyte, the analyte may provide one or more coordination atomsfor the metal, effectively replacing the small polar ligands, due totheir good leaving group properties or poor sigma donor properties.These ligands may be referred to as “substitutionally labile”. Theligands preferably form dipoles, since this will contribute to a highsolvent reorganization energy.

[0031] Irreversible ligand groups include, but are not limited to,amines (—NH₂, —NHR, and —NR₂, with R being a substitution group that ispreferably small and hydrophilic, as will be appreciated by those in theart), cyano groups (—C═N), thiocyano groups (—SC═N), and isothiocyanogroups (—N═CS). Reversible ligand groups include, but are not limitedto, H₂O and halide atoms or groups. It should be understood that thechange in solvent reorganization energy is quite high when a watermolecule serves as a coordination atom; thus, the replacement oraddition of a single water molecule on a redox active molecule willgenerally result in a detectable change, even when the other ligands arenot small polar ligands. Thus, in a preferred embodiment, the inventionrelies on the replacement or addition of at least one water molecule ona redox active molecule.

[0032] In addition to small polar ligands, the metal ions may haveadditional, hydrophobic ligands, also depicted herein as “L”. That is, ahexacoordinate metal ion such as Fe may have one ligand position(preferably axial) filled by the spacer used for attachment to theelectrode, two ligand positions filled by phenanthroline, and two orthree small polar ligands, depending on the linkage to the bindingligand. As appreciated by those in the art, a wide variety of suitableligands may be used. Suitable traditional ligands include, but are notlimited to, isonicotinamide; imidazole; bipyridine and substitutedderivatives of bipyridine; terpyridine and substituted derivatives;phenanthrolines, particularly 1,10-phenanthroline (abbreviated phen) andsubstituted derivatives of phenanthrolines such as4,7-dimethylphenanthroline and dipyridol[3,2-a:2′,3′-c]phenazine(abbreviated dppz); dipyridophenazine; 1,4,5,8,9,12-hexaazatriphenylene(abbreviated hat); 9,10-phenanthrenequinone diimine (abbreviated phi);1,4,5,8-tetraazaphenanthrene (abbreviated tap); 1,4,8,11-tetra-azacyclotetradecane (abbreviated cyclam), isocyanide andmetallocene ligands. Substituted derivatives, including fusedderivatives, may also be used.

[0033] The presence of at least one small, polar ligand on thetransition metal complex makes the solvent reorganization energy high,which suppresses electron transfer to and from the transition metalredox active molecule. Thus, in a preferred embodiment, a solventaccessible redox active molecule has a solvent reorganization energy ofgreater than about 500 meV, with greater than about 800 meV beingpreferred, greater than about 1 eV being especially preferred andgreater than about 1.2 to 1.3 eV being particularly preferred.

[0034] In addition to the solvent accessible redox active molecule, aredox active complex comprises a binding ligand which will bind thetarget analyte. By “binding ligand” or grammatical equivalents herein ismeant a compound that is used to probe for the presence of the targetanalyte, and that will specifically bind to the analyte; the bindingligand is part of a binding pair. By “specifically bind” herein is meantthat the ligand binds the analyte, with specificity sufficient todifferentiate between the analyte and other components or contaminantsof the test sample. This binding should be sufficient to remain boundunder the conditions of the assay, including wash steps to removenon-specific binding. Generally, the disassociation constants of theanalyte to the binding ligand will be in the range of at least 10⁻⁴-10⁻⁶M⁻¹, with a preferred range being 10⁻⁵ to 10⁻⁹ M⁻¹ and a particularlypreferred range being 10⁻⁷-10⁻⁹ M⁻¹.

[0035] As will be appreciated by those in the art, the composition ofthe binding ligand will depend on the composition of the target analyte.Binding ligands to a wide variety of analytes are known or can bereadily found using known techniques. For example, when the analyte is asingle-stranded nucleic acid, the binding ligand may be a complementarynucleic acid. Alternatively, the binding ligand may be a nucleicacid-binding protein when the analyte is a single or double-strandednucleic acid. When the analyte is a protein, the binding ligands includeproteins or small molecules. Preferred binding ligand proteins includepeptides. For example, when the analyte is an enzyme, suitable bindingligands include substrates and inhibitors. Antigen-antibody pairs,receptor-ligands, and carbohydrates and their binding partners are alsosuitable analyte-binding ligand pairs.

[0036] In general, preferred embodiments utilize relatively smallbinding ligands and larger target analytes.

[0037] Together, the transition metal complex and the binding ligandcomprise a redox active complex. In addition, there may be more than onebinding ligand or transition metal complex per redox active complex. Theredox active complex may also contain additional moieties, such ascross-linking agents, labels, etc., and linkers for attachment to theelectrode.

[0038] The redox active complex is bound to an electrode. This may beaccomplished in any number of ways, as will be apparent to those in theart. Generally, as is more fully described below, one or both of thetransition metal complex and the binding ligand are attached, via aspacer, to the electrode.

[0039] In a preferred embodiment, the redox active complex is covalentlyattached to the electrode via a spacer. By “spacer” herein is meant amoiety which holds the redox active complex off the surface of theelectrode. In a preferred embodiment, the spacer is a conductiveoligomer as described herein, although suitable spacer moieties includepassivation agents and insulators as outlined below. The spacer moietiesmay be substantially non-conductive, although preferably (but notrequired) is that the electron coupling between the redox activemolecule and the electrode (H_(AB)) does not become the rate limitingstep in electron transfer.

[0040] In general, the length of the spacer is as described forconductive polymers and passivation agents. As will be appreciated bythose in the art, if the spacer becomes too long, the electroniccoupling between the redox active molecule and the electrode willdecrease.

[0041] In a preferred embodiment, the spacer is a conductive oligomer.By “conductive oligomer” herein is meant a substantially conductingoligomer, preferably linear, some embodiments of which are referred toin the literature as “molecular wires”. Conductive oligomers, and theirsynthesis, use and attachment to moieties is described in PCTUS97/20014, hereby expressly incorporated in its entirety.

[0042] By “substantially conducting” herein is meant that the electroncoupling between the transition metal complex and the electrode (H_(AB))throught the oligomer is not the rate limiting step of electrontransfer. Generally, the conductive oligomer has substantiallyoverlapping π-orbitals, i.e. conjugated π-orbitals, as between themonomeric units of the conductive oligomer, although the conductiveoligomer may also contain one or more sigma (σ) bonds. Additionally, aconductive oligomer may be defined functionally by its ability to passelectrons into or from an attached transition metal complex.Furthermore, the conductive oligomer is more conductive than theinsulators as defined herein.

[0043] In a preferred embodiment, the conductive oligomers have aconductivity, S, of from between about 10⁻⁶ to about 10⁴ Ω⁻¹cm⁻¹, withfrom about 10⁻⁵ to about 10³ Ω⁻¹cm⁻¹ being preferred, with these Svalues being calculated for molecules ranging from about 20 Å to about200 Å. As described below, insulators have a conductivity S of about10⁻⁷ Ω⁻¹cm⁻¹ or lower, with less than about 10⁻⁸ Ω⁻¹cm⁻¹ beingpreferred. See generally Gardner et al., Sensors and Actuators A 51(1995) 57-66, incorporated herein by reference.

[0044] Desired characteristics of a conductive oligomer include highconductivity, sufficient solubility in organic solvents and/or water forsynthesis and use of the compositions of the invention, and preferablychemical resistance to reactions that occur i) during synthesis of theredox active complexes, ii) during the attachment of the conductiveoligomer to an electrode, or iii) during analyte assays.

[0045] The oligomers of the invention comprise at least two monomericsubunits, as described herein. As is described more fully below,oligomers include homo- and hetero-oligomers, and include polymers.

[0046] In a preferred embodiment, the conductive oligomer has thestructure depicted in Structure 1:

[0047] As will be understood by those in the art, all of the structuresdepicted herein may have additional atoms or structures; i.e. theconductive oligomer of Structure 1 may be attached to transition metalcomplexes or redox active complexes, binding ligands, electrodes, etc.or to several of these. Unless otherwise noted, the conductive oligomersdepicted herein will be attached at the left side to an electrode; thatis, as depicted in Structure 1, the left “Y” is connected to theelectrode as described herein and the right “Y”, if present, is attachedto the redox active complex, i.e. either the transition metal complex orbinding ligand, either directly or through the use of a linker, as isdescribed herein

[0048] In this embodiment, Y is an aromatic group, n is an integer from1 to 50, g is either 1 or zero, e is an integer from zero to 10, and mis zero or 1. When g is 1, B-D is a bond able to conjugate withneighboring bonds (herein referred to as a “conjugated bond”),preferably selected from acetylene, alkene, substituted alkene, amide,azo, —C═N— (including —N═C—, —CR═N— and —N═CR—), —Si═Si—, and —Si═C—(including —C═Si—, —Si═CR— and —CR═Si—). When g is zero, e is preferably1, D is preferably carbonyl, or a heteroatom moiety, wherein theheteroatom is selected from oxygen, sulfur, nitrogen or phosphorus.Thus, suitable heteroatom moieties include, but are not limited to, —NHand —NR, wherein defined herein; substituted sulfur; sulfonyl (—SO₂—)sulfoxide (—SO—); phosphine oxide (—PO— and —RPO—); and thiophosphine(—PS— and —RPS—). However, when the conductive oligomer is to beattached to a gold electrode, as outlined below, sulfur derivatives arenot preferred.

[0049] By “aromatic group” or grammatical equivalents herein is meant anaromatic monocyclic or polycyclic hydrocarbon moiety generallycontaining 5 to 14 carbon atoms (although larger polycyclic ringsstructures may be made) and any carbocylic ketone or thioketonederivative thereof, wherein the carbon atom with the free valence is amember of an aromatic ring. Aromatic groups include arylene groups andaromatic groups with more than two atoms removed. For the purposes ofthis application aromatic includes heterocycle. “Heterocycle” or“heteroaryl” means an aromatic group wherein 1 to 5 of the indicatedcarbon atoms are replaced by a heteroatom chosen from nitrogen, oxygen,sulfur, phosphorus, boron and silicon wherein the atom with the freevalence is a member of an aromatic ring, and any heterocyclic ketone andthioketone derivative thereof. Thus, heterocycle includes thienyl,furyl, pyrrolyl, pyrimidinyl, oxalyl, indolyl, purinyl, quinolyl,isoquinolyl, thiazolyl, imidozyl, etc.

[0050] Importantly, the Y aromatic groups of the conductive oligomer maybe different, i.e. the conductive oligomer may be a heterooligomer. Thatis, a conductive oligomer may comprise an oligomer of a single type of Ygroups, or of multiple types of Y groups. Thus, in a preferredembodiment, when a barrier monolayer is used as is described below, oneor more types of Y groups are used in the conductive oligomer within themonolayer with a second type(s) of Y group used above the monolayerlevel. Thus, as is described herein, the conductive oligomer maycomprise Y groups that have good packing efficiency within the monolayerat the electrode surface, and a second type(s) of Y groups with greaterflexibility and hydrophilicity above the monolayer level to facilitatetarget analyte binding. For example, unsubstituted benzyl rings maycomprise the Y rings for monolayer packing, and substituted benzyl ringsmay be used above the monolayer. Alternatively, heterocylic rings,either substituted or unsubstituted, may be used above the monolayer.Additionally, in one embodiment, heterooligomers are used even when theconductive oligomer does not extend out of the monolayer.

[0051] The aromatic group may be substituted with a substitution group,generally depicted herein as R. R groups may be added as necessary toaffect the packing of the conductive oligomers, i.e. when the conductiveoligomers form a monolayer on the electrode, R groups may be used toalter the association of the oligomers in the monolayer. R groups mayalso be added to 1) alter the solubility of the oligomer or ofcompositions containing the oligomers; 2) alter the conjugation orelectrochemical potential of the system; and 3) alter the charge orcharacteristics at the surface of the monolayer.

[0052] Suitable R groups include, but are not limited to, hydrogen,alkyl, alcohol, aromqtic, amino, amido, nitro, ethers, esters,aldehydes, ketones, iminos, sulfonyl, silicon moieties, halogens, sulfurcontaining moieties, phosphorus containing moieties, and ethyleneglycols. In the structures depicted herein, P is hydrogen when theposition is unsubstituted. It should be noted that some positions mayallow two substitution groups, R and R′, in which case the R and R′groups may be either the same or different.

[0053] By “alkyl group” or grammatical equivalents herein is meant astraight or branched chain alkyl group, with straight chain alkyl groupsbeing preferred. If branched, it may be branched at one or morepositions, and unless specified, at any position. The alkyl group mayrange from about 1 to about 30 carbon atoms (C1-C30), with a preferredembodiment utilizing from about 1 to about 20 carbon atoms (C1-C20),with about C1 through about C12 to about C15 being preferred, and C1 toC5 being particularly preferred, although in some embodiments the alkylgroup may be much larger. Also included within the definition of analkyl group are cycloalkyl groups such as C5 and C6 rings, andheterocyclic rings with nitrogen, oxygen, sulfur, silicon or phosphorus.Alkyl also includes heteroalkyl, with heteroatoms of sulfur, oxygen,nitrogen, and silicone being preferred. Alkyl includes substituted alkylgroups. By “substituted alkyl group” herein is meant an alkyl groupfurther comprising one or more substitution moieties “R”, as definedabove.

[0054] By “amino groups” or grammatical equivalents herein is meant—NH₂, —NHR and —NR₂ groups, with R being as defined herein.

[0055] By “nitro group” herein is meant an —NO₂ group.

[0056] By “sulfur containing moieties” herein is meant compoundscontaining sulfur atoms, including but not limited to, thia-, thio- andsulfo- compounds, thiols (—SH and —SR), sulfides (—RSR—), sulfoxides(—R—SO—R—), sulfones (—R—SO₂—R—), disulfides (—R—S—S—R—) and sulfonylester (R—SO₂—O—R) groups. By “phosphorus containing moieties” herein ismeant compounds containing phosphorus, including, but not limited to,phosphines and phosphates. By “silicon containing moieties” herein ismeant compounds containing silicon, including siloxanes.

[0057] By “ether” herein is meant an —O—R group.

[0058] By “ester” herein is meant a —COOR group; esters includethioesters (—CSOR).

[0059] By “halogen” herein is meant bromine, iodine, chlorine, orfluorine. Preferred substituted alkyls are partially or fullyhalogenated alkyls such as CF₃, etc.

[0060] By “aldehyde” herein is meant —RCOH groups.

[0061] By “ketone” herein is meant —R—CO—R groups.

[0062] By “alcohol” herein is meant —OH groups, and alkyl alcohols —ROH.

[0063] By “amido” herein is meant —RCONH— or RCONR— groups.

[0064] By “imino” herein is meant and —R—CNH—R— and —R—CNR—R— groups.

[0065] By “ethylene glycol” herein is meant a —(O—CH₂—CH₂)_(n)— group,although each carbon atom of the ethylene group may also be singly ordoubly substituted, i.e. —(O—CR₂—CR₂)_(n)—, with R as described above.Ethylene glycol derivatives with other heteroatoms in place of oxygen(i.e. —(N—CH₂—CH₂)_(n)— or —(S—CH₂—CH₂)_(n)—, or with substitutiongroups) are also preferred.

[0066] Preferred substitution groups include, but are not limited to,methyl, ethyl, propyl, and ethylene glycol and derivatives thereof.

[0067] Preferred aromatic groups include, but are not limited to,phenyl, naphthyl, naphthalene, anthracene, phenanthroline, pyrole,pyridine, thiophene, porphyrins, and substituted derivatives of each ofthese, included fused ring derivatives.

[0068] In the conductive oligomers depicted herein, when g is 1, B-D isa bond linking two atoms or chemical moieties. In a preferredembodiment, B-D is a conjugated bond, containing overlapping orconjugated π-orbitals.

[0069] Preferred B-D bonds are selected from acetylene (—C═C—, alsocalled alkyne or ethyne), alkene (—CH═CH—, also called ethylene),substituted alkene (—CR═CR—, —CH═CR— and —CR═CH—), amide (—NH—CO— and—NR—CO— or —CO—NH— and —CO—NR—), azo (—N═N—), esters and thioesters(—CO—O—, —O—CO—, —CS—O— and —O—CS—) and other conjugated bonds such as(—CH═N—, —CR═N—, —N═CH— and —N═CR—), (—SiH═SiH—, —SiR═SiH—, —SiR═SiH—,and —SiR═SiR—), (—SiH═CH—, —SiR═CH—, —SiH═CR—, —SiR═CR—, —CH═SiH—,—CR═SiH—, —CH═SiR—, and —CR═SiR—). Particularly preferred B-D bonds areacetylene, alkene, amide, and substituted derivatives of these three,and azo. Especially preferred B-D bonds are acetylene, alkene and amide.The oligomer components attached to double bonds may be in the trans orcis conformation, or mixtures. Thus, either B or D may include carbon,nitrogen or silicon. The substitution groups are as defined as above forR.

[0070] When g=0 in the Structure 1 conductive oligomer, e is preferably1 and the D moiety may be carbonyl or a heteroatom moiety as definedabove.

[0071] As above for the Y rings, within any single conductive oligomer,the B-D bonds (or D moieties, when g=0) may be all the same, or at leastone may be different. For example, when m is zero, the terminal B-D bondmay be an amide bond, and the rest of the B-D bonds may be acetylenebonds. Generally, when amide bonds are present, as few amide bonds aspossible are preferable, but in some embodiments all the B-D bonds areamide bonds. Thus, as outlined above for the Y rings, one type of B-Dbond may be present in the conductive oligomer within a monolayer asdescribed below, and another type above the monolayer level, to givegreater flexibility for nucleic acid hybridization.

[0072] In the structures depicted herein, n is an integer from 1 to 50,although longer oligomers may also be used (see for example Schumm etal., Angew. Chem. Int. Ed. Engl. 1994 33(13):1360). Without being boundby theory, it appears that for efficient binding of target analytes, thebinding should occur at a distance from the surface. For example, itappears that the kinetics of nucleic acid hybridization increase as afunction of the distance from the surface, particularly for longoligonucleotides of 200 to 300 basepairs. Accordingly, the length of theconductive oligomer is such that the binding ligand is positioned fromabout 6 Å to about 100 Å (although distances of up to 500 Å may be used)from the electrode surface, with from about 25 Å to about 60 Å beingpreferred. Accordingly, n will depend on the size of the aromatic group,but generally will be from about 1 to about 20, with from about 2 toabout 15 being preferred and from about 3 to about 10 being especiallypreferred.

[0073] In the structures depicted herein, m is either 0 or 1. That is,when m is 0, the conductive oligomer may terminate in the B-D bond or Dmoiety, i.e. the D atom is attached to the redox active complex ormolecule, or binding ligand, either directly or via a linker. In someembodiments there may be additional atoms, such as a linker, attachedbetween the conductive oligomer and the bound moiety. Alternatively,when m is 1, the conductive oligomer may terminate in Y, an aromaticgroup, i.e. the aromatic group is attached to the moiety or linker.

[0074] As will be appreciated by those in the art, a large number ofpossible conductive oligomers may be utilized. These include conductiveoligomers falling within the Structure 1 and Structure 4 formulas, aswell as other conductive oligomers, as are generally known in the art,including for example, compounds comprising fused aromatic rings orTeflon®-like oligomers, such as —(CF₂)_(n)—, —(CHF)_(n)— and—(CFR)_(n)—. See for example, Schumm et al., Angew. Chem. Intl. Ed.Engl. 33:1361 (1994); Grosshenny et al., Platinum Metals Rev.40(1):26-35 (1996); Tour, Chem. Rev. 96:537-553 (1996); Hsung et al.,Organometallics 14:48084815 (1995; and references cited therein, all ofwhich are expressly incorporated by reference.

[0075] Particularly preferred conductive oligomers of this embodimentare depicted below:

[0076] Structure 2 is Structure 1 when g is 1. Preferred embodiments ofStructure 2 include: e is zero, Y is pyrole or substituted pyrole; e iszero, Y is thiophene or substituted thiophene; e is zero, Y is furan orsubstituted furan; e is zero, Y is phenyl or substituted phenyl; e iszero, Y is pyridine or substituted pyridine; e is 1, B-D is acetyleneand Y is phenyl or substituted phenyl. A preferred embodiment ofStructure 2 is also when e is one, depicted as Structure 3 below:

[0077] Preferred embodiments of Structure 3 are: Y is phenyl orsubstituted phenyl and B-D is azo; Y is phenyl or substituted phenyl andB-D is alkene; Y is pyridine or substituted pyridine and B-D isacetylene; Y is thiophene or substituted thiophene and B-D is acetylene;Y is furan or substituted furan and B-D is acetylene; Y is thiophene orfuran (or substituted thiophene or furan) and B-D are alternating alkeneand acetylene bonds.

[0078] In a preferred embodiment, the conductive oligomer has thestructure depicted in Structure 4:

[0079] In this embodiment, C are carbon atoms, n is an integer from 1 to50, m is 0 or 1, J is a heteroatom selected from the group consisting ofnitrogen, silicon, phosphorus, sulfur, carbonyl or sulfoxide, and G is abond selected from alkane, alkene or acetylene, such that together withthe two carbon atoms the C'G—C group is an alkene (—CH═CH—), substitutedalkene (—CR═CR—) or mixtures thereof (—CH═CR— or —CR═CH—), acetylene(—C═C—), or alkane (—CR₂—CR₂—, with R being either hydrogen or asubstitution group as described herein). The G bond of each subunit maybe the same or different than the G bonds of other subunits; that is,alternating oligomers of alkene and acetylene bonds could be used, etc.However, when G is an alkane bond, the number of alkane bonds in theoligomer should be kept to a minimum, with about six or less sigma bondsper conductive oligomer being preferred. Alkene bonds are preferred, andare generally depicted herein, although alkane and acetylene bonds maybe substituted in any structure or embodiment described herein as willbe appreciated by those in the art.

[0080] In a preferred embodiment, the m of Structure 4 is zero. In aparticularly preferred embodiment, m is zero and G is an alkene bond, asis depicted in Structure 5:

[0081] The alkene oligomer of structure 5, and others depicted herein,are generally depicted in the preferred trans configuration, althougholigomers of cis or mixtures of trans and cis may also be used. Asabove, R groups may be added to alter the packing of the compositions onan electrode, the hydrophilicity or hydrophobicity of the oligomer, andthe flexibility, i.e. the rotational, torsional or longitudinalflexibility of the oligomer. n is as defined above.

[0082] In a preferred embodiment, R is hydrogen, although R may be alsoalkyl groups and polyethylene glycols or derivatives.

[0083] In an alternative embodiment, the conductive oligomer may be amixture of different types of oligomers, for example of Structures 1 and4.

[0084] The conductive oligomers are covalently attached to the redoxactive complexes, transition metal complexes (collectively redox activemoieties), or binding ligands. By “covalently attached” herein is meantthat two moieties are attached by at least one bond, including sigmabonds, pi bonds and coordination bonds.

[0085] The redox active moiety or binding ligand is covalently attachedto the conductive oligomer, and the conductive oligomer is alsocovalently attached to the electrode. In general, the covalentattachments are done in such a manner as to minimize the amount ofunconjugated sigma bonds an electron must travel from the electron donorto the electron acceptor. Thus, linkers are generally short, or containconjugated bonds with few sigma bonds.

[0086] The covalent attachment of the redox active moiety or bindingligand and the conductive oligomer may be accomplished in a variety ofways, and will depend on the composition of the redox active moiety orbinding ligand, as will be appreciated by those in the art.Representative conformations of the attachment of redox active complexesto electrodes are depicted below in Structures 6 and 7:

[0087] In Structure 6, the hatched marks on the left represent anelectrode. X is a conductive oligomer as defined herein. F₁ is a linkagethat allows the covalent attachment of the electrode and the conductiveoligomer, including bonds, atoms or linkers such as are describedherein. F₂ is a linkage that allows the covalent attachment of theconductive oligomer to the redox active complex, which includes thebinding ligand, BL. F₁ and F₂ may be a bond, an atom or a linkage as isherein described. F₂ may be part of the conductive oligomer, part of theredox active complex, or exogeneous to both. As for Structure 7, M isthe metal ion and L is a co-ligand, as defined herein; as noted above,if a traditional hydrophobic ligand is used, two or more of the depictedL ligands may be part of multidentate ligand, rather than separateligands. It should be noted that while the BL is depicted in an axialposition, this is not required.

[0088] In this embodiment, a coordination atom may be contributeddirectly from the binding ligand; alternatively, there may be a linkerthat provides a coordination atom and is linked to the binding ligand. Awide variety of linkers can be used herein, as will be appreciated bythose in the art. Suitable linkers are known in the art, for example,homo-or hetero-bifunctional linkers as are well known (see 1994 PierceChemical Company catalog, technical section on cross-linkers, pages155-200, incorporated herein by reference). Preferred linkers include,but are not limited to, alkyl groups and alkyl groups containingheteroatom moieties, with short alkyl groups, esters, epoxy groups andethylene glycol and derivatives being preferred, with propyl, acetylene,and C₂ alkene being especially preferred.

[0089] Structure 7 depicts a “branched” conformation, wherein thetransition metal complex and the binding ligand are not directlyattached. As will be appreciated by those in the art, the transitionmetal complex and the binding ligand may be attached at the sameposition on the conductive oligomer, or different positions, and morethan one transition metal complex and/or binding ligand may be present.

[0090] Structures 6 and 7 depict hexacoordinate metal ions, although aswill be appreciated by those in the art, other types of metal ions alsofind use in the invention, with the appropriate adjustment of L ligands.

[0091] The attachment of the metal ion is generally done by attaching asubstitutionally inert ligand to the end of the spacer. In a preferredembodiment, this ligand is monodentate, or at most bidentate, althoughother polydentate ligands may also be used. Thus, for example, an aminoor imidazole group (monodentate) or a phenathroline (bidentate) may beattached to the end of the spacer using techniques well known in theart, or techniques outlined in PCT US97/20014, hereby expresslyincorporated by reference.

[0092] The attachment of the binding ligand to either the metal ion orthe spacer is also done using well known techniques, and will depend onthe composition of the binding ligand. When the binding ligand is anucleic acid, either double-stranded or single-stranded, attachment tothe metal ion can be done as is described in PCT US97/20014.

[0093] In general, attachment of the binding ligand to either the metalion or the spacer is done using functional groups either naturally foundon the binding ligand or added using well known techniques. These groupscan be at the terminus of the binding ligand, for example at the N- orC-terminus of a protein, or at any internal position. Thus, amino, thio,carboxyl or amido groups can all be used for attachment. Similarly,chemical attachment of traditional ligands such as pyridine orphenanthroline may also be done, as will be appreciated by those in theart. For example, attachment of proteinaceous binding ligands isgenerally done using functional groups present on the amino acid sidechains or at the N- or C-terminus; for example, any groups such as theN-terminus or side chains such as histidine may serve as ligands for themetal ion. Similarly, attachment of carbohydrate binding ligands isgenerally done by derivatizing the sugar to serve as a metal ion ligand.Alternatively, these groups may be used to attach to the spacer, usingwell known techniques. In any of these embodiments, there may beadditional connector or linkers present. For example, when the bindingligand is a proteinaceous enzyme substrate or inhibitor, there may beadditional amino acids, or an alkyl group, etc., between the metal ionligand and the functional substrate or inhibitor.

[0094] In addition, as noted herein, two or more binding ligands may beattached to a single redox active complex. For example, twosingle-stranded nucleic acids may be attached, such that the binding ofa complementary target sequence will change the solvent reorganizationenergy of the redox active molecule. In this embodiment, the two singlestranded nucleic acids are designed to allow for a “gap” in thecomplementary sequence to accomodate the metal ion; this is generallyfrom 1 to 3 nucleotides.

[0095] In a preferred embodiment, the binding ligand and the redoxactive molecule do not form a redox active complex, but rather are eachindividually attached to the electrode, generally via a spacer. In thisembodiment, it is the proximity of the redox active molecule to thetarget analyte bound to the binding ligand that results in a decrease ofthe solvent reorganization energy upon binding. Preferably, the solventaccessible redox active molecule is within 12 Å of some portion of thetarget analyte, with less than about 8 Å being preferred and less thanabout 5 Å being particularly preferred, and less than about 3.5 Å beingespecially preferred. It should be noted that the distance between thebinding ligand and the redox active molecule may be much larger,depending on the size of the target analyte. Thus, the binding of alarge target analyte may reduce the solvent reorganization energy of asolvent accessible redox active molecule many angstroms away from thebinding ligand. A representative composition is depicted below inStructure 8:

[0096] In Structure 8, the binding ligand and the transition metalcomplex are separately attached to the electrode. While Structure 8depicts a 1:1 ratio of transition metal complexes to binding ligands,this is not required; in fact, it may be preferable to have an excess oftransition metal complexes on the electrode, particularly when thetarget analyte is relatively large in comparison to the transition metalcomplex. Thus, for example, a single binding event of a target analyteto a binding ligand can result in a decrease in solvent reorganizationenergy for a number of transition metal complexes, if the density of thetransition metal complexes is high enough in the area of the bindingligand, or the target analyte is large enough. Similarly, differentbinding ligands for the same target analyte may be used; for example, to“tack down” a large target analyte on the surface, to effect as manytransition metal complexes as possible per single target analyte.

[0097] The redox moieties and binding ligands are attached to anelectrode, via a spacer as outlined above. Thus, one end or terminus ofthe conductive oligomer is attached to the redox moiety or bindingligand, and the other is attached to an electrode. In some embodimentsit may be desirable to have the conductive oligomer attached at aposition other than a terminus, or to have a branched conductiveoligomer that is attached to an electrode at one terminus and to a redoxactive molecule and a binding ligand at other termini. Similarly, theconductive oligomer may be attached at two sites to the electrode.

[0098] By “electrode” herein is meant a composition, which, whenconnected to an electronic device, is able to sense a current or chargeand convert it to a signal. Preferred electodes are known in the art andinclude, but are not limited to, certain metals and their oxides,including gold; platinum; palladium; silicon; aluminum; metal oxideelectrodes including platinum oxide, titanium oxide, tin oxide, indiumtin oxide, palladium oxide, silicon oxide, aluminum oxide, molybdenumoxide (Mo₂O₆), tungsten oxide (WO₃) and ruthenium oxides; and carbon(including glassy carbon electrodes, graphite and carbon paste).Preferred electrodes include gold, silicon, carbon and metal oxideelectrodes.

[0099] The electrodes described herein are depicted as a flat surface,which is only one of the possible conformations of the electrode and isfor schematic purposes only. The conformation of the electrode will varywith the detection method used. For example, flat planar electrodes maybe preferred for optical detection methods, or when arrays are made,thus requiring addressable locations for both synthesis and detection.Alternatively, for single analyte analysis, the electrode may be in theform of a tube, with the compositions of the invention bound to theinner surface. This allows a maximum of surface area containing thebinding ligand to be exposed to a small volume of sample.

[0100] The covalent attachment of the conductive oligomer containing theredox active moieties and binding ligands of the invention may beaccomplished in a variety of ways, depending on the electrode and theconductive oligomer used. Generally, some type of linker is used. Forexample, in Structure 6, F₁ may be a linker or atom. The choice of “F₁”will depend in part on the characteristics of the electrode. Thus, forexample, F₁ may be a sulfur moiety when a gold electrode is used.Alternatively, when metal oxide electrodes are used, F₁ may be a silicon(silane) moiety attached to the oxygen of the oxide (see for exampleChen et al., Langmuir 10:3332-3337 (1994); Lenhard et al., J.Electroanal. Chem. 78:195-201 (1977), both of which are expresslyincorporated by reference). When carbon based electrodes are used, F₁may be an amino moiety (preferably a primary amine; see for exampleDeinhammer et al., Langmuir 10:1306-1313 (1994)). Thus, preferred F₁moieties include, but are not limited to, silane moieties, sulfurmoieties (including alkyl sulfur moieties), and amino moieties. In apreferred embodiment, epoxide type linkages with redox polymers such asare known in the art are not used.

[0101] Although depicted herein as a single moiety, the conductiveoligomer may be attached to the electrode with more than one F₁ moiety;the F₁ moieties may be the same or different. Thus, for example, whenthe electrode is a gold electrode, and F₁ is a sulfur atom or moiety,multiple sulfur atoms may be used to attach the conductive oligomer tothe electrode. Preferably, the F₁ moiety is just a sulfur atom, butsubstituted sulfur moieties may also be used.

[0102] In a preferred embodiment, the electrode is a gold electrode, andattachment is via a sulfur linkage as is well known in the art, i.e. theF₁ moiety is a sulfur atom or moiety. Although the exact characteristicsof the gold-sulfur attachment are not known, this linkage is consideredcovalent for the purposes of this invention.

[0103] In a preferred embodiment, the electrode is a carbon electrode,i.e. a glassy carbon electrode, and attachment is via a nitrogen of anamine group.

[0104] In general, one of two general schemes may be followed tosynthesize the compositions of the invention. In a preferred embodiment,the spacer is synthesized and the redox active complex, comprising theredox active molecule and the binding ligand is also made separately.These two are added together, and then added to the electrode.Alternatively, in a preferred embodiment, the spacer is made andattached to the electrode. The redox active complex is made, and then itis added to the spacer. General synthetic schemes may be found in PCTUS97/20014.

[0105] Thus, in a preferred embodiment, electrodes are made thatcomprise conductive oligomers attached to redox active moieties and/orbinding ligands for the purposes of analyte assays, as is more fullydescribed herein. As will be appreciated by those in the art, electrodescan be made that have a single species of binding ligand (i.e. specificfor a particular analyte) or multiple binding ligand species (i.e.multiple analytes).

[0106] In addition, as outlined herein, the use of a solid support suchas an electrode enables the use of these binding ligands in an arrayform. The use of arrays of binding ligands specific for oligonucleotidesare well known in the art. In addition, techniques are known for“addressing” locations within an electrode and for the surfacemodification of electrodes.

[0107] Thus, in a preferred embodiment, arrays of different bindingligands are laid down on the electrode, each of which are covalentlyattached to the electrode via a conductive linker. In this embodiment,the number of different species of binding ligands may vary widely, fromone to thousands, with from about 4 to about 100,000 being preferred,and from about 10 to about 10,000 being particularly preferred.

[0108] In a preferred embodiment, the electrode further comprises apassivation agent, preferably in the form of a monolayer on theelectrode surface. For some analytes, such as nucleic acids, theefficiency of analyte binding (i.e. hybridization) may increase when thebinding ligand is at a distance from the electrode. In addition, thepresence of a monolayer can decrease non-specific binding to thesurface. A passivation agent layer facilitates the maintenance of thebinding ligand and/or analyte away from the electrode surface. Inaddition, a passivation agent serves to keep charge carriers away fromthe surface of the electrode. Thus, this layer helps to preventelectrical contact between the electrodes and the electron transfermoieties, or between the electrode and charged species within thesolvent. Such contact can result in a direct “short circuit” or anindirect short circuit via charged species which may be present in thesample. Accordingly, the monolayer of passivation agents is preferablytightly packed in a uniform layer on the electrode surface, such that aminimum of “holes” exist. Alternatively, the passivation agent may notbe in the form of a monolayer, but may be present to help the packing ofthe conductive oligomers or other characteristics.

[0109] The passivation agents thus serve as a physical barrier to blocksolvent accesibility to the electrode. As such, the passivation agentsthemselves may in fact be either (1) conducting or (2) nonconducting,i.e. insulating, molecules. Thus, in one embodiment, the passivationagents are conductive oligomers, as described herein, with or without aterminal group to block or decrease the transfer of charge to theelectrode. Other passivation agents which may be conductive includeoligomers of —(CF₂)_(n)—, —(CHF)_(n)— and —(CFR)_(n)—. In a preferredembodiment, the passivation agents are insulator moieties.

[0110] An “insulator” is a substantially nonconducting oligomer,preferably linear. By “substantially nonconducting” herein is meant thatthe rate of electron transfer through the insulator is slower than therate of electron transfer through the a conductive oligomer. Stateddifferently, the electrical resistance of the insulator is higher thanthe electrical resistance of the conductive oligomer. It should be notedhowever that even oligomers generally considered to be insulators, suchas —(CH₂)₁₆ molecules, still may transfer electrons, albeit at a slowrate.

[0111] In a preferred embodiment, the insulators have a conductivity, S,of about 10⁻⁷ Ω⁻¹cm⁻¹ or lower, with less than about 10⁻⁸ Ω⁻¹cm⁻¹ beingpreferred. See generally Gardner et al., supra.

[0112] Generally, insulators are alkyl or heteroalkyl oligomers ormoieties with sigma bonds, although any particular insulator moleculemay contain aromatic groups or one or more conjugated bonds. By“heteroalkyl” herein is meant an alkyl group that has at least oneheteroatom, i.e. nitrogen, oxygen, sulfur, phosphorus, silicon or boronincluded in the chain. Alternatively, the insulator may be quite similarto a conductive oligomer with the addition of one or more heteroatoms orbonds that serve to inhibit or slow, preferably substantially, electrontransfer.

[0113] The passivation agents, including insulators, may be substitutedwith R groups as defined herein to alter the packing of the moieties orconductive oligomers on an electrode, the hydrophilicity orhydrophobicity of the insulator, and the flexibility, i.e. therotational, torsional or longitudinal flexibility of the insulator. Forexample, branched alkyl groups may be used. In addition, the terminus ofthe passivation agent, including insulators, may contain an additionalgroup to influence the exposed surface of the monolayer. For example,the addition of charged, neutral or hydrophobic groups may be done toinhibit non-specific binding from the sample, or to influence thekinetics of binding of the analyte, etc. For example, there may benegatively charged groups on the terminus to form a charged surface suchthat when the nucleic acid is DNA or RNA the nucleic acid is repelled orprevented from lying down on the surface.

[0114] The length of the passivation agent will vary as needed.Generally, the length of the passivation agents is similar to the lengthof the conductive oligomers, as outlined above. In addition, theconductive oligomers may be basically the same length as the passivationagents or longer than them, resulting in the binding ligands being moreaccessible to the solvent.

[0115] The monolayer may comprise a single type of passivation agent,including insulators, or different types.

[0116] Suitable insulators are known in the art, and include, but arenot limited to, —(CH₂)_(n)—, —(CRH)_(n)—, and —(CR₂)_(n)—, ethyleneglycol or derivatives using other heteroatoms in place of oxygen, i.e.nitrogen or sulfur (sulfur derivatives are not preferred when theelectrode is gold).

[0117] The passivation agents are generally attached to the electrode inthe same manner as the conductive oligomer, and may use the same linkeras defined above.

[0118] The target analyte, contained within a test sample, is added tothe electrode containing either a solvent accessible redox activecomplex or a mixture of solvent accessible transition metal complexesand binding ligands, under conditions that if present, the targetanalyte will bind to the binding ligand. These conditions are generallyphysiological conditions. Generally a plurality of assay mixtures arerun in parallel with different concentrations to obtain a differentialresponse to the various concentrations. Typically, one of theseconcentrations serves as a negative control, i.e., at zero concentrationor below the level of detection. In addition, any variety of otherreagents may be included in the screening assay. These include reagentslike salts, neutral proteins, e.g. albumin, detergents, etc which may beused to facilitate optimal binding and/or reduce non-specific orbackground interactions. Also reagents that otherwise improve theefficiency of the assay, such as protease inhibitors, nucleaseinhibitors, anti-microbial agents, etc., may be used. The mixture ofcomponents may be added in any order that provides for the requisitebinding.

[0119] In a preferred embodiment, the target analyte will bind thebinding ligand reversibly, i.e. non-covalently, such as inprotein-protein interactions of antigens-antibodies, enzyme-substrate(or some inhibitors) or receptor-ligand interactions.

[0120] In a preferred embodiment, the target analyte will bind thebinding ligand irreversibly, for example covalently. For example, someenzyme-inhibitor interactions are considered irreversible.Alternatively, the analyte initially binds reversibly, with subsequentmanipulation of the system which results in covalent attachment. Forexample, chemical cross-linking after binding may be done, as will beappreciated by those in the art. For example, peptides may becross-linked using a variety of bifunctional agents, such asmaleimidobenzoic acid, methyidithioacetic acid, mercaptobenzoic acid,S-pyridyl dithiopropionate, etc. Alternatively, functionally reactivegroups on the target analyte and the binding ligand may be induced toform covalent attachments.

[0121] Upon binding of the analyte to the binding moiety, the solventaccessible transition metal complex becomes solvent inhibited. By“solvent inhibited transition metal complex” herein is meant the solventreorganization energy of the solvent inhibited transition metal complexis less than the solvent reorganization energy of the solvent accessibletransition metal complex. As noted above, this may occur in severalways. In a preferred embodiment, the target analyte provides acoordination atom, such that the solvent accessible transition metalcomplex loses at least one, and preferably several, of its small polarligands. Alternatively, in a preferred embodiment, the proximity of thetarget analyte to the transition metal complex does not result in ligandexchange, but rather excludes solvent from the area surrounding themetal ion (i.e. the first or second coordination sphere) thuseffectively lowering the required solvent reorganization energy.

[0122] In a preferred embodiment, the required solvent reorganizationenergy decreases sufficiently to result in a decrease in the E₀ of theredox active molecule by at about 100 mV, with at least about 200 mVbeing preferred, and at least about 300-500 mV being particularlypreferred.

[0123] In a preferred embodiment, the required solvent reorganizationenergy decreases by at least 100 mV, with at least about 200 mV beingpreferred, and at least about 300 -500 mV being particularly preferred.

[0124] In a preferred embodiment, the the required solventreorganization energy decreases sufficiently to result in a rate changeof electron transfer (kET) between the solvent inhibited transitionmetal complex and the electrode relative to the rate of electrontransfer between the solvent accessible transition metal complex and theelectrode. In a preferred embodiment, this rate change is greater thanabout a factor of 3, with at least about a factor of 10 being preferredand at least about a factor of 100 or more being particularly preferred.

[0125] The determination of solvent reorganization energy will be doneas is appreciated by those in the art. Briefly, as outlined in Marcustheory, the electron transfer rates (k_(ET)) are determined at a numberof different driving forces (or free energy, −ΔG°); the point at whichthe rate equals the free energy is the activationless rate (A). This maybe treated in most cases as the equivalent of the solvent reorganizationenergy; see Gray et al. Ann. Rev. Biochem. 65:537 (1996), herebyincorporated by reference.

[0126] The solvent inhibited transition metal complex, indicating thepresence of a target analyte, is detected by intiating electron transferand detecting a signal characteristic of electron transfer between thesolvent inhibited redox active molecule and the electrode.

[0127] Electron transfer is generally initiated electronically, withvoltage being preferred. A potential is applied to a sample containingmodified nucleic acid probes. Precise control and variations in theapplied potential can be via a potentiostat and either a three electrodesystem (one reference, one sample and one counter electrode) or a twoelectrode system (one sample and one counter electrode). This allowsmatching of applied potential to peak electron transfer potential of thesystem which depends in part on the choice of transition metal complexesand in part on the conductive oligomer used.

[0128] Preferably, initiation and detection is chosen to maximize therelative difference between the solvent reorganization energies of thesolvent accessible and solvent inhibited transition metal complexes.

[0129] Electron transfer between the transition metal complex and theelectrode can be detected in a variety of ways, with electronicdetection, including, but not limited to, amperommetry, voltammetry,capacitance and impedance being preferred. These methods include time orfrequency dependent methods based on AC or DC currents, pulsed methods,lock-in techniques, and filtering (high pass, low pass, band pass). Insome embodiments, all that is required is electron transfer detection;in others, the rate of electron transfer may be determined.

[0130] In a preferred embodiment, electronic detection is used,including amperommetry, voltammetry, capacitance, and impedance.Suitable techniques include, but are not limited to, electrogravimetry;coulometry (including controlled potential coulometry and constantcurrent coulometry); voltametry (cyclic voltametry, pulse voltametry(normal pulse voltametry, square wave voltametry, differential pulsevoltametry, Osteryoung square wave voltametry, and coulostatic pulsetechniques); stripping analysis (aniodic stripping analysis, cathiodicstripping analysis, square wave stripping voltammetry); conductancemeasurements (electrolytic conductance, direct analysis); time-dependentelectrochemical analyses (chronoamperometry, chronopotentiometry, cyclicchronopotentiometry and amperometry, AC polography, chronogalvametry,and chronocoulometry); AC impedance measurement, capacitancemeasurement; AC voltametry, and photoelectrochemistry.

[0131] In a preferred embodiment, monitoring electron transfer is viaamperometric detection. This method of detection involves applying apotential (as compared to a separate reference electrode) between theelectrode containing the compositions of the invention and an auxiliary(counter) electrode in the test sample. Electron transfer of differingefficiencies is induced in samples in the presence or absence of targetanalyte.

[0132] The device for measuring electron transfer amperometricallyinvolves sensitive current detection and includes a means of controllingthe voltage potential, usually a potentiostat. This voltage is optimizedwith reference to the potential of the redox active molecule.

[0133] In a preferred embodiment, alternative electron detection modesare utilized. For example, potentiometric (or voltammetric) measurementsinvolve non-faradaic (no net current flow) processes and are utilizedtraditionally in pH and other ion detectors. Similar sensors are used tomonitor electron transfer between the redox active molecules and theelectrode. In addition, other properties of insulators (such asresistance) and of conductors (such as conductivity, impedance andcapicitance) could be used to monitor electron transfer between theredox active molecules and the electrode. Finally, any system thatgenerates a current (such as electron transfer) also generates a smallmagnetic field, which may be monitored in some embodiments.

[0134] In a preferred embodiment, the system may be calibrated todetermine the amount of solvent accessible transition metal complexes onan electrode by running the system in organic solvent prior to theaddition of target. This is quite significant to serve as an internalcontrol of the sensor or system. This allows a preliminary measurement,prior to the addition of target, on the same molecules that will be usedfor detection, rather than rely on a similar but different controlsystem. Thus, the actual molecules that will be used for the detectioncan be quantified prior to any experiment Running the system in theabsence of water, i.e. in organic solvent such as acetonitrile, willexclude the water and substantially negate any solvent reorganizationeffects. This will allow a quantification of the actual number ofmolecules that are on the surface of the electrode. The sample can thenbe added, an output signal determined, and the ratio of bound/unboundmolecules determined. This is a significant advantage over priormethods.

[0135] It should be understood that one benefit of the fast rates ofelectron transfer observed in the compositions of the invention is thattime resolution can greatly enhance the signal-to-noise results ofmonitors based on electronic current The fast rates of electron transferof the present invention result both in high signals and stereotypeddelays between electron transfer initiation and completion. Byamplifying signals of particular delays, such as through the use ofpulsed initiation of electron transfer and “lock-in” amplifiers ofdetection, orders of magnitude improvements in signal-to-noise may beachieved.

[0136] Without being bound by theory, it appears that target analytes,bound to an electrode, may respond in a manner similar to a resistor andcapacitor in series. Also, the E₀ of the redox active molecule can shiftas a result of the target analyte binding. Furthermore, it may bepossible to distinguish between solvent accessible and solvent inhibitedtransition metal complexes on the basis of the rate of electrontransfer, which in turn can be exploited in a number of ways fordetection of the target analyte. Thus, as will be appreciated by thosein the art, any number of initiation-detection systems can be used inthe present invention.

[0137] In a preferred embodiment, electron transfer is initiated anddetected using direct current (DC) techniques. As noted above, the E₀ ofthe redox active molecule can shift as a result of the change in thesolvent reorganization energy upon target analyte binding. Thus,measurements taken at the E₀ of the solvent accessible transition metalcomplex and at the E₀ of the solvent inhibited complex will allow thedetection of the analyte. As will be appreciated by those in the art, anumber of suitable methods may be used to detect the electron transfer.

[0138] In a preferred embodiment, electron transfer is initiated usingalternating current (AC) methods. A first input electrical signal isapplied to the system, preferably via at least the sample electrode(containing the complexes of the invention) and the counter electrode,to initiate electron transfer between the electrode and the secondelectron transfer moiety. Three electrode systems may also be used, withthe voltage applied to the reference and working electrodes. In thisembodiment, the first input signal comprises at least an AC component.The AC component may be of variable amplitude and frequency. Generally,for use in the present methods, the AC amplitude ranges from about 1 mVto about 1.1 V, with from about 10 mV to about 800 mV being preferred,and from about 10 mV to about 500 mV being especially preferred. The ACfrequency ranges from about 0.01 Hz to about 10 MHz, with from about 1Hz to about 1 MHz being preferred, and from about 1 Hz to about 100 kHzbeing especially preferred

[0139] In a preferred embodiment, the first input signal comprises a DCcomponent and an AC component. That is, a DC offset voltage between thesample and counter electrodes is swept through the electrochemicalpotential of the electron transfer moiety. The sweep is used to identifythe DC voltage at which the maximum response of the system is seen. Thisis generally at or about the electrochemical potential of the transitionmetal complex. Once this voltage is determined, either a sweep or one ormore uniform DC offset voltages may be used. DC offset voltages of fromabout −1 V to about +1.1 V are preferred, with from about −500 mV toabout +800 mV being especially preferred, and from about −300 mV toabout 500 mV being particularly preferred. On top of the DC offsetvoltage, an AC signal component of variable amplitude and frequency isapplied. If the transition metal complex has a low enough solventreorganization energy to respond to the AC perturbation, an AC currentwill be produced due to electron transfer between the electrode and thetransition metal complex.

[0140] In a preferred embodiment, the AC amplitude is varied. Withoutbeing bound by theory, it appears that increasing the amplitudeincreases the driving force. Thus, higher amplitudes, which result inhigher overpotentials give faster rates of electron transfer. Thus,generally, the same system gives an improved response (i.e. higheroutput signals) at any single frequency through the use of higheroverpotentials at that frequency. Thus, the amplitude may be increasedat high frequencies to increase the rate of electron transfer throughthe system, resulting in greater sensitivity. In addition, as notedabove, it may be possible to distinguish between solvent accessible andsolvent inhibited transition metal complexes on the basis of the rate ofelectron transfer, which in turn can be used either to distinguish thetwo on the basis of frequency or overpotential.

[0141] In a preferred embodiment, measurements of the system are takenat at least two separate amplitudes or overpotentials, with measurementsat a plurality of amplitudes being preferred. As noted above, changes inresponse as a result of changes in amplitude may form the basis ofidentification, calibration and quantification of the system.

[0142] In a preferred embodiment, the AC frequency is varied. Atdifferent frequencies, different molecules respond in different ways. Aswill be appreciated by those in the art, increasing the frequencygenerally increases the output current. However, when the frequency isgreater than the rate at which electrons may travel between theelectrode and the transition metal complexes, higher frequencies resultin a loss or decrease of output signal. At some point, the frequencywill be greater than the rate of electron transfer through even solventinhibited transition metal complexes, and then the output signal willalso drop.

[0143] In addition, the use of AC techniques allows the significantreduction of background signals at any single frequency due to entitiesother than the target analyte, i.e. “locking out” or “filtering”unwanted signals. That is, the frequency response of a charge carrier orredox active species in solution will be limited by its diffusioncoefficient. Accordingly, at high frequencies, a charge carrier may notdiffuse rapidly enough to transfer its charge to the electrode, and/orthe charge transfer kinetics may not be fast enough. This isparticularly significant in embodiments that do not utilize apassivation layer monolayer or have partial or insufficient monolayers,i.e. where the solvent is accessible to the electrode. As outlinedabove, in DC techniques, the presence of “holes” where the electrode isaccessible to the solvent can result in solvent charge carriers “shortcircuiting” the system. However, using the present AC techniques, one ormore frequencies can be chosen that prevent a frequency response of oneor more charge carriers in solution, whether or not a monolayer ispresent. This is particularly significant since many biological fluidssuch as blood contain significant amounts of redox active species whichcan interfere with amperometric detection methods.

[0144] In a preferred embodiment, measurements of the system are takenat at least two separate frequencies, with measurements at a pluralityof frequencies being preferred. A plurality of frequencies includes ascan. In a preferred embodiment, the frequency response is determined atat least two, preferably at least about five, and more preferably atleast about ten frequencies.

[0145] After transmitting the input signal to initiate electrontransfer, an output signal is received or detected. The presence andmagnitude of the output signal will depend on theoverpotential/amplitude of the input signal; the frequency of the inputAC signal; the composition of the intervening medium, i.e. theimpedance, between the electron transfer moieties; the DC offset; theenvironment of the system; and the solvent. At a given input signal, thepresence and magnitude of the output signal will depend in general onthe solvent reorganization energy required to bring about a change inthe oxidation state of the metal ion. Thus, upon transmitting the inputsignal, comprising an AC component and a DC offset, electrons aretransferred between the electrode and the transition metal complex, whenthe solvent reorganization energy is low enough, the frequency is inrange, and the amplitude is sufficient, resulting in an output signal.

[0146] In a preferred embodiment, the output signal comprises an ACcurrent. As outlined above, the magnitude of the output current willdepend on a number of parameters. By varying these parameters, thesystem may be optimized in a number of ways.

[0147] In general, AC currents generated in the present invention rangefrom about 1 femptoamp to about 1 milliamp, with currents from about 50femptoamps to about 100 microamps being preferred, and from about 1picoamp to about 1 microamp being especially preferred.

[0148] In addition, those in the art will appreciate that it is alsopossible to use the compositions of the invention in assays that rely ona loss of signal. For example, a first measurement is taken when thetransition metal complex is inhibited, and then the system is changed asa result of the introduction of a target analyte, causing the solventinhibited molecule to become solvent accessible, resulting in a loss ofsignal. This may be done in several ways, as will be appreciated bythose in the art.

[0149] In a preferred embodiment, a first measurement is taken when thetarget analyte is present. The target analyte is then removed, forexample by the use of high salt concentrations or thermal conditions,and then a second measurement is taken. The quantification of the lossof the signal can serve as the basis of the assay.

[0150] Alternatively, the target analyte may be an enzyme. In thispreferred embodiment, the transition metal complex is made solventinhibited by the presence of an enzyme substrate or analog, preferably,but not required to be covalently attached to the transition metalcomplex, preferably as one or more ligands. Upon introduction of thetarget enzyme, the enzyme associates with the substrate to cleave orotherwise sterically alter the substrate such that the transition metalcomplex is made solvent accessible. This change can then be detected.This embodiment is advantageous in that it results in an amplificationof the signal, since a single enzyme molecule can result in multiplesolvent accessible molecules. This may find particular use in thedetection of bacteria or other pathogens that secrete enzymes,particularly scavenger proteases or carbohydrases.

[0151] Similarly, a preferred embodiment utilizes competition-typeassays. In this embodiment, the binding ligand is the same as the actualmolecule for which detection is desired; that is, the binding ligand isactually the target analyte or an analog. A binding partner of thebinding ligand is added to the surface, such that the transition metalcomplex becomes solvent inhibited, electron transfer occurs and a signalis generated. Then the actual test sample, containing the same orsimilar target analyte which is bound to the electrode, is added. Thetest sample analyte will compete for the binding partner, causing theloss of the binding partner on the surface and a resulting decrease inthe signal.

[0152] A similar embodiment utilizes a target analyte (or analog) iscovalently attached to a preferably larger moiety (a “blocking moiety”).The analyte-blocking moiety complex is bound to a binding ligand thatbinds the target analyte, serving to render the transition metal complexsolvent inhibited. The introduction of the test sample target analyteserves to compete for the analyte-blocking moiety complex, releasing thelarger complex and resulting in a more solvent accessible molecule.

[0153] In addition, while the majority of the above discussion isdirected to the use of the invention when the compositions are attachedto surfaces such as electrodes, those of skill in the art willappreciate that solution-based systems are also possible. In thisembodiment, solvent accessible transition metal complexes are attachedto binding ligands (either directly or using short linkers that keep thebinding ligand and the transition metal complex in close enoughproximity to allow detection) to form soluble redox active complexes.Upon binding of an analyte, the transition metal complex becomes solventinhibited, and a change in the system can be detected. In a preferredembodiment, the reaction is monitored by fluorescence or electrochemicalmeans. Alternatively, the reaction may be monitored electronically,using mediators.

[0154] The present invention further provides apparatus for thedetection of analytes using AC detection methods. The apparatus includesa test chamber which has at least a first measuring or sample electrode,and a second measuring or counter electrode. Three electrode systems arealso useful. The first and second measuring electrodes are in contactwith a test sample receiving region, such that in the presence of aliquid test sample, the two electrodes may be in in electrical contact.

[0155] In a preferred embodiment, the first measuring electrodecomprises a redox active complex, covalently attached via a spacer, andpreferably via a conductive oligomer, such as are described herein.Alternatively, the first measuring electrode comprises covalentlyattached transition metal complexes and binding ligands.

[0156] The apparatus further comprises a voltage source electricallyconnected to the test chamber; that is, to the measuring electrodes.Preferably, the voltage source is capable of delivering AC and DCvoltages, if needed.

[0157] In a preferred embodiment, the apparatus further comprises aprocessor capable of comparing the input signal and the output signal.The processor is coupled to the electrodes and configured to receive anoutput signal, and thus detect the presence of the target analyte.

[0158] The compositions of the present invention may be used in avariety of research, clinical, quality control, or field testingsettings.

[0159] The following examples serve to more fully describe the manner ofusing the above-described invention, as well as to set forth the bestmodes contemplated for carrying out various aspects of the invention. Itis understood that these examples in no way serve to limit the truescope of this invention, but rather are presented for illustrativepurposes. All references cited herein are incorporated by reference intheir entireity.

EXAMPLE Preparation of Solvent Accessible Redox Active Moiety

[0160] In this example, a solution based sensor was made, using a redoxactive complex comprising a ruthenium complex and a biotin bindingligand. A transition metal complex of ruthenium, with small polarcoordination ligands (NH₃), was made. One of the coordination atoms wasprovided by the binding ligand norbiotin (biotin conjugated with aprimary amine via four carbon linker), such that upon binding of avidin,the transition metal complex goes from solvent accessible to solventinhibited. This was detected fluorometrically. Alternatively, the redoxactive complex of ruthenium and biotin can be activated and added to asurface to form an electrode-based sensor.

[0161] The synthesis of [trans Rull (NH₃)₄(norbiotin) Cl]Cl₁₂ wascarried in several steps. The first intermediate in the reactionsequence is trans-[SO₂ (NH₃) RulllCl]Cl] and was synthesized in thefollowing manner.

[0162] 2.5 gr. (8.5 mmoles) of [(NH₃)₅ RulllCl]Cl₂ was slurried in 65 mlof pre-heated water (˜70° C.) in a three neck round bottom flaskequipped with a thermometer, reflux condenser and gas inlet. To thisflask was added 3.55 gr (2.4 mmoles) of NaHSO₃ and immediately acontinuous stream of SO gas was bubbled through the solution and themixture allowed to warm to 83° C. The reaction was allowed to proceedfor 90 minutes. The solution was cooled to 0° C., and the productcollected and washed several times with acetone.

[0163] The solid was slurried in 200 mls of 6M HCl and heated to avigorous reflux for 20 min in a 500 ml flask. The reaction mixture wasfiltered and allowed to stand at 4° C. overnight. The rust coloredcrystals of trans-[SO(NH₃) RullCl]Cl were collected and slurried in 50ml of water, heated to 40° C., an excess of norbiotin was added and thesolution allowed to react for 30 minutes. The solution was transferredto a 1000 ml flask and 750 ml of acetone was added and allowed to stirfor 10 minutes. The solid was collected, washed with acetone and driedin vacuo.

[0164] The solid was dissolved in a minimum of water and filtered. Tothis solution was added dropwise with stirring a 50:50 mixture of 30%H₂O₂ and 2N HCl. A solid was obtained by the addition of 15 volumes ofacetone, collected and dried in vacuo. This product was dissolved in aminimum amount of degassed 0.15N HCl and thoroughly degassed. Zinc-Hgamalgam was prepared, the solution was transferred to the zinc amalgam,and the reaction allowed to proceed for 1 hour. A previously degassedsolution of 1M BaCl₂ was added.

[0165] The solid, including the amalgam, was filtered as quickly aspossible into a filter flask containing 3-4 ml of 30% H₂O₂ and 3M HCl.The product was obtained from the yellow solution via precipitationusing 15 volumes of acetone, collected, washed and dried in vacuo. Thesolid was redissolved in a minimum amount of 0.01 N HCl and applied to a4×30 cm column of SP Sephadex C-25. The product was recovered using 0.2NHCl.

[0166] The collected fractions were evaporated to dryness, dissolved ina minimum amount of 0.01 N HCl, filtered and precipitated with 15volumes of acetone. The product [trans Rulll (NH₃)₄(norbiotin) Cl]CI₂was collected, and dried in vacuo.

[0167] For a solution-based sensor, the material was taken up in waterand a fluorescence measurement was taken; the sample exhibited nofluorescence; that is, the presence of water was, a barrier tofluorescence. Avidin was added and a second fluorescence measurement wastaken; in the presence of avidin, fluorescence was detected. This showsthat the environment around the complex is altered such that the wateris no longer a barrier to fluorescence; i.e. fluorescence is notquenched.

[0168] For an electrode-based sensor, the material can be activated foraddition to a conductive oligomer as follows. The [trans Rulll(NH₃)₄(norbiotin) Cl]Cl₂ is activated by reduction of the complex usingZinc-Hg amalgam to form [trans Rull (NH₃)₄(norbiotin) H₂O]Cl₂ underinert atmosphere conditions. To this material is added a conductiveoligomer that terminates in a group suitable to serve as a coordinationatom, such as a nitrogen-containing species, such as analine. Theconductive oligomer containing the redox active complex (i.e. thesolvent accessible transition metal complex and the binding ligand) canthen be mixed with other monolayer-forming components such aspassivation agents and added to the electrode using known techniques,such as those described in PCT US97/20014, hereby incorporated byreference.

I claim:
 1. A composition comprising an electrode with a covalentlyattached redox active complex comprising a binding ligand and a solventaccessible transition metal complex.
 2. A composition according to claim1 wherein said solvent accessible transition metal complex has at leasttwo coordination sites occupied by polar coordination groups.
 3. Acomposition according to claim 1 wherein said solvent accessibletransition metal complex has at least one coordination site occupied bya water molecule.
 4. A composition according to claim 1 wherein saidelectrode further comprises a self-assembled monolayer.
 5. A compositionaccording to claim I wherein said solvent accessible transition metalcomplex is covalently attached to said electrode via a conductiveoligomer.
 6. A composition according to claim 1 wherein said solventaccessible transition metal complex is linked to said binding ligand toform a redox active complex.
 7. A composition according to claim 1wherein said binding ligand is covalently attached to said electrode viaa conductive oligomer.
 8. A method according to claim 1, wherein saidsolvent accessible transition metal complex has a solvent reorganizationenergy of greater than about 1200 mV.
 9. A method of detecting a targetanalyte in a test sample comprising: a) binding an analyte to a redoxactive complex comprising: i) a solvent accessible transition metalcomplex having at least one coordination site occupied by a polarcoordination group; and ii) a binding ligand that will bind the targetanalyte; wherein said redox active complex is bound to an electrode,such that upon binding, a solvent inhibited transition metal complex isformed; and b) detecting electron transfer between said solventinhibited transition metal complex and said electrode.
 10. A methodaccording to claim 9, wherein said solvent accessible transition metalcomplex has a solvent reorganization energy of greater than about 1200mV and said solvent inhibited transition metal complex has a solventreorganization energy of less than 1000 mV.
 11. A method according toclaim 9, wherein the solvent reorganization energy of said solventinhibited transition metal complex decreases by at least 100 mV uponbinding of said analyte to form said solvent inhibited transition metalcomplex.
 12. A method according to claim 9, wherein upon binding, atleast one solvent accessible transition metal complex is less than 8 Åfrom the bound analyte such that it forms said solvent inhibitedtransition metal complex.
 13. A method according to claim 9, whereinsaid polar coordination group is a water molecule.
 14. A methodaccording to claim 9 further comprising applying at least a first inputsignal to said solvent inhibited transition metal complex.
 15. A methodaccording to claim 14 wherein in the absence of target analyte, saidfirst input signal does not result in significant electron transfer. 16.A method according to claim 14, wherein said first input signalcomprises at least an AC component.
 17. A method according to claim 14further comprising applying input signal at a plurality of frequencies.18. A method according to claim 14, wherein said first input signalcomprises at least a DC voltage.
 19. A method according to claim 18further comprising applying input signal at a plurality of voltages. 20.A method according to claim 9 wherein said detecting is by receiving anoutput signal characteristic of electron transfer between said solventinhibited transition metal complex and said electrode.
 21. A methodaccording to claim 20 wherein said output signal is a current.
 22. Amethod according to claim 21 wherein said current is an AC current. 23.A method according to claim 9, wherein said binding ligand is covalentlyattached to said solvent accessible transition metal complex.
 24. Amethod according to claim 9, wherein said ligand is covalently attachedto said electrode.
 25. A method according to claim 9, wherein saidsolvent accessible transition metal complex is covalently attached tosaid electrode.
 26. A method according to claim 25 wherein said covalentattachment is via a conductive oligomer.
 27. A method according to claim9, wherein said analyte is a biomolecule.
 28. A method according toclaim 27, wherein said biomolecule is selected from the group consistingof proteins, carbohydrates, and lipids.
 29. An apparatus for thedetection of target analytes in a test sample, comprising: a) a testchamber comprising a first and a second measuring electrode, whereinsaid first measuring electrode comprises a covalently attached redoxactive complex comprising: i) a solvent accessible transition metalcomplex having at least one coordination site occupied by a polarcoordination group; and ii) a binding ligand; b) an AC/DC voltage sourceelectrically connected to said test chamber.
 30. An apparatus accordingto claim 29 wherein said covalent attachment is via a spacer.
 31. Anapparatus according to claim 29 further comprising a processor coupledto said electrodes.
 32. An apparatus according to claim 29 wherein saidelectrode further comprises a self-assembled monolayer.